Male Wistar rats (age, 65–80 days; weight, 250–350 g) were used. The animals were housed in plastic cages with food and water available ad libitum, under a 12 h light/dark cycle (lights on at 7:00 A.M.) at a constant temperature of 22 ± 1°. The animal sample size for each experiment was determined on the basis of our experience (Sacco and Sacchetti, 2010; Cambiaghi et al., 2016a, b) and of the current literature. All experiments were conducted in accordance with the European Communities Council 2010/63/EU and approved by the Italian Ministry of Health (Authorization No. 265/2011) and by the local Bioethical Committee of the University of Turin.

Reversible functional inactivation of the secondary auditory cortex Te2 was induced by the bilateral administration of Tetrodotoxin citrate (TTX) at the following stereotaxic coordinates: anteroposterior (AP), −5.8; lateral (L), ±6.5; and ventral (V), 6.0, 0.5 μl; and (AP), −6.8; (L), ±6.5 and (V), 6.0, 0.55 μl (Cambiaghi et al., 2016b). TTX (10 ng/μl) was dissolved in saline and then injected 24 h after training. Control subjects received saline solution instead of TTX. Rats were mounted in the stereotaxic apparatus (ear bars, 45°), an incision of the skull was made, and small burr holes were drilled to allow the penetration of a 28 gauge infusion needle. A 10 μl Hamilton syringe mounted on an infusion pump was used to deliver infusions at a rate of 0.25 μl/min. The needle was left in place for another 1 min. Incisions were closed (stainless steel wound clips), and animals were given s.c. injections of the analgesic/anti-inflammatory ketoprofen (2 mg/kg). Rats were kept warm and under observation until recovery from anesthesia.

For recording of extracellular field potentials, stainless steel wires were implanted unilaterally (right side) at least 1 week before memory recall. Electrodes were built with three stainless steel wires (Ø 150 μm) to ensure mechanical stability of the bundle (to obtain straight trajectory in the brain tissue), and they were connected to a 10-pin connector (Omnetics). Under deep anesthesia, electrodes were stereotaxically implanted in BLA and Te2, according to the following coordinates, in mm: BLA, AP = −2.7, L = 5.0, V = 8.1; Te2, AP = −6.6, L = 6.5–6.7, V = 6.0. A silver wire over contralateral parietal areas served as reference and ground. All implants were secured using Ketacem cement. All recordings were performed in a customized Faraday chamber. Local field potentials (LFPs) were recorded (Plexon acquisition system, 16-channel) and initially digitalized at 1 kHz and stored on a hard drive for offline analysis. The LFPs were very similar across the three channels belonging to the same bundle. At the conclusion of each experiment, animals were transcardially perfused for the detection of the recording sites throughout Nissl staining.

LFPs epochs were visually examined and power spectra of artifact-free segments were computed using fast Fourier transforms by using the commercial software NeuroExplorer with a 0.25 Hz resolution. Mean power spectra were divided into five frequency bands: delta (0.5–3 Hz), low-theta (3.01–7 Hz), high-theta (7.01–12 Hz), beta1 (12.01–20 Hz) and beta2 (20.01–30 Hz). Baseline was evaluated by averaging three 2-s epochs within the pre-CS period. For the 8 s analysis we averaged four windows of 2 s length with 0% overlap. Relative power was calculated by dividing the absolute amplitude within the aforementioned frequency ranges by the corresponding measures of total amplitude. Spectrograms were calculated using the software NeuroExplorer.

The coherence between LFP channels was measured by magnitude squared coherence (MSC), using the function mscohere in Matlab signal toolbox, which is a coherence estimate of the input signals x and y by using Welch’s averaged, modified periodogram method. The MSC estimate is a function of frequency with values between 0 and 1 and indicates how well x corresponds to y at each frequency. Segments of 2 s duration are split into 8 epochs with 50% overlap. The MSC estimate is calculated over the frequency range of 0.5–30 Hz for each rat. Difference in coherence were obtained by subtraction of coherence values (CS−preCS) and statistics were performed on the average difference in coherence within the frequency bands of interest. PreCS was calculated by averaging five 2 s epochs within the entire pre-CS period (Cambiaghi et al., 2016a).

As in our previous study (Sacco and Sacchetti, 2010), 4 weeks after training, all animals were handled and habituated to the new cage for 5 days, each day for 7 min. The cage was different from that employed during conditioning. The sixth day, all groups were exposed to the acoustic stimuli (see “fear memory retention” paragraph). Freezing was used as an index to measure fear conditioning. Ninety minutes after the completion of memory retention test, rats were deeply anesthetized and perfused intracardially with 4% paraformaldehyde. The brains were dissected, stored overnight at 4°C, and finally transferred to 30% sucrose. Coronal sections (50-μm) were cut on a vibratome and collected in PBS. Free-floating sections were pretreated with 0.3% H₂O₂ in PBS to reduce endogenous peroxidase activity. After four rinses, sections were incubated in a blocking solution (2% bovine serum albumin (BSA), 2% normal goat serum and 0.2% Triton X-100) for 1 h at RT). Then, they were incubated in primary polyclonal rabbit anti-zif268 (1:2000 dilution, Santa Cruz) antibodies in the blocking solution overnight al the RT. Subsequently, sections were washed with PBS and incubated for 2 h at RT with biotinylated goat antirabbit IgG (1:2000 in PBS, Jackson Laboratories) followed by 1 h at RT in ABC. Sections were rinsed in PBS. The peroxidase reaction end-product was visualized by incubating sections in 0.05 M Tris (pH 7.6) containing 3.3′ DAB (0.037%) as chromogen and hydrogen peroxide (0.015%) for 5 min. Finally, immunolabeled sections were washed in PBS, mounted on gelatin-coated slides, dehydrated and coverslipped. The slices were analyzed using Neurolucida software connected to a microscope via a color CCD camera. The quantification of zif268-positive cells was carried out at X 10 magnification. Immunoreactive nuclei were counted bilaterally using at least three serial sections for each area without experimenter knowledge of the experimental condition. The number of nuclei expressing zif268 was quantified in the area of interest at the coordinates: lateral and basal nuclei of the amygdala AP = from −2.5 mm to −3.3 mm; central amygdala AP = from −2.0 mm to −2.8 mm (Sacco and Sacchetti, 2010). The mean count of each animal was divided by the mean count of the respective naïve control group in order to generate a normalized count for each animal. Data were then averaged in order to produce the mean of each group.

The needle track in the case of TTX-injections was histologically verified at the end of the experiments with Nissl staining, using the conventional procedure (Sacco and Sacchetti, 2010).

Student’s t-test was used for comparing freezing responses. One-way ANOVA and Newman-Keuls multiple comparisons test were employed to compare Zif268 protein levels in the different behavioral groups. Nonparametric Kruskal-Wallis test followed by Wilcoxon signed-rank test were employed for the electrophysiological analysis. Experiments were replicated in two (for electrophysiological recordings) and in three (for immunohistochemical analysis) independent trials. All results were reported as means with Standard Error Mean (SEM) as indicated in figure legends.

To establish the effects that Te2 blockade performed during the consolidation of auditory fear memories may have on Te2-to-BLA crosstalk on BLA activity occurring during memory retrieval process, we reversibly blocked the Te2 at 1 day after training and we subsequently tested long-term memory retention and Te2-BLA interplay 1 month later (Figures 1, 2A). As in our previous studies (Grosso et al., 2015b; Cambiaghi et al., 2016a), in order to reversibly block Te2 during memory consolidation processes, we administered Tetrodotoxin (TTX), a voltage-dependent sodium channels blocker. Unlike optogenetic manipulations or muscimol, TTX blocks neural activity for several hours (i.e., for at least 6–8 h; Zhuravin and Bures, 1991; Ambrogi Lorenzini et al., 1999; Martin and Ghez, 1999), and it is therefore suitable for interfering with long-term memory consolidation processes that require fast synaptic transmission for several hours and days (Ambrogi Lorenzini et al., 1999; Riedel et al., 1999; Sacchetti et al., 1999a, 2002; Lesburguères et al., 2011). Moreover, TTX has fully reversible effects and does not induce any permanent damage (Ambrogi Lorenzini et al., 1999; Sacchetti et al., 1999a, 2002). To interfere with long-term system consolidation but not with the cellular consolidation mechanisms that are triggered immediately after training, TTX was administered 1 day after learning (Lesburguères et al., 2011; Cambiaghi et al., 2016b).

Rats were trained to associate acoustic stimuli CSs with aversive ones USs. Long-term memory retention was assessed 1 month later by measuring freezing behavior elicited by CSs previously paired with the US (Sacchetti et al., 1999b; Sacco and Sacchetti, 2010; Grosso et al., 2015b). In line with our previous studies (Grosso et al., 2015b; Cambiaghi et al., 2016a), freezing was significantly lower in TTX-treated animals (n = 9) compared to control conditioned ones (n = 12), while it was similar between TTX-treated rats and unpaired (n = 10) animals (Figure 1A). As in our previous study (Cambiaghi et al., 2016a), in order to investigate between Te2 and BLA during memory retrieval, we analyzed LFP in Te2 and BLA. An interval of 2 s at the onset of the first CS was analyzed for each animal in conditioned, TTX-injected and unpaired rats (Figure 2B). In fact, our previous study showed that Te2-to-BLA synchronization occurred during the initial 2 s at the onset of the first CS, whilst it was not present the last 2 s of the first CS presentation (Cambiaghi et al., 2016a). By comparing the coherence during the CS onset with respect to the preCS, we found higher levels of coherence in the conditioned group in the low-theta (3.01–7 Hz) range (Kruskal-Wallis test, P = 0.011), but not in the other frequency bands namely delta (0.5–3 Hz; Kruskal-Wallis test, P = 0.982), high-theta (7.01–12 Hz; Kruskal-Wallis test, P = 0.098), beta1 (12.01–20 H; Kruskal-Wallis test, P = 0.473) and beta2 (20.01–30 Hz; Kruskal-Wallis test, P = 0.653) ranges (Figures 2C–F). In animals retrieving long-term memories, the coherence between Te2 and BLA in the low-theta range was significantly enhanced (median, 0.206) with respect to unpaired (median, −0.024; Mann Whitney test, P = 0.006) animals. Conversely, in TTX-conditioned rats, there was no difference in coherence (median, −0.037) from unpaired rats (P = 0.388), while coherence was significantly lower in TTX-conditioned rats than the conditioned group (P = 0.022; Figures 2C,D). These findings show that Te2 blockade upon memory consolidation processes prevented the theta synchrony occurring between this cortex and the BLA during long-term memory retrieval.

We then investigated the effects that blocking Te2 upon memory consolidation could have on the amygdala activity during the retrieval of fearful memories. The neural activity of the BLA was analyzed during retrieval of long-term fear memories in conditioned rats, in those treated with TTX and in unpaired animals. Initially, we analyzed the early phase of memory retrieval (first 2 s), but in order to detect any possible change in activity that could emerge after the cue recall in both Te2 and BLA, we also investigated the entire first CS (8 s). Relative power was analyzed in both Te2 and BLA by dividing the absolute amplitude within the frequency bands delta (0.5–3 Hz), low-theta (3.01–7 Hz), high-theta (7.01–12 Hz), beta1 (12.01–20 Hz) and beta2 (20.01–30 Hz) by the corresponding measures of total amplitude during the pre-CS period (Figures 3A,B). In animals retrieving long-term memories, the early recall phase (2 s) showed significant differences in Te2 (Figure 3C) and BLA (Figure 3D) LFP powers within both the low- and high-theta frequency bands (Kruskal-Wallis, P = 0.002 and P = 0.015 for BLA, respectively; P = 0.009 and P = 0.037 for Te2, respectively), but not in the delta, beta1 or beta 2 bands (Kruskal-Wallis, P = 0.334, P = 0.168 and P = 0.482 for BLA, respectively; P = 0.384, P = 0.735 and P = 0.553 for Te2, respectively). However, TTX-injected rats showed no change in the low theta range with respect to unpaired rats in both Te2 (Figure 3C; Mann Whitney test, P = 0.706 and P = 0.179 respectively) and BLA (Figure 3D; Mann Whitney test, P = 0.094 and P = 0.053 respectively). The increased activity observed in the 7–12 Hz range in unpaired vs. TTX-Te2 rats might be due to non-associative fear related processes.

These data were obtained by analyzing neural activity during the initial 2 s of the first CS and therefore showed that TTX administration into Te2 impaired early memory-related processes during the initial stages of memory retrieval in both Te2 and BLA. However, during the retrieval of the overall learned experience, BLA may display a learning-evoked change due to information encoded in brain sites other than the Te2. To address this issue, we analyzed LFPs of Te2 and BLA during the entire duration (8 s) of the first CS (Figures 4A–D). In both Te2 and BLA, we found a significant difference within the low-theta range among the three groups (Kruskal-Wallis, P = 0.001 and P = 0.031, respectively), with conditioned rats showing the highest levels of power with respect to both unpaired and TTX-Te2 groups (Mann Whitney test, P < 0.05 in both instances; Figures 4E,F). No differences were detected among the three groups in the high-theta band (Kruskal-Wallis, P = 0.468 and P = 0.914, respectively; Figures 4E,F). In the Te2, a significant difference was observed in the delta range (Kruskal-Wallis, P = 0.046), in which conditioned rats had an increased power with respect to both the other groups (Mann Whitney test, P < 0.05 in both instances; Figure 4E). In addition, in BLA we found that even in the 12–20 Hz range (Kruskal-Wallis, P = 0.034) conditioned rats presented an increased power with respect to unpaired and TTX-Te2 groups (Mann Whitney test, P = 0.029 and P = 0.027, respectively; Figure 4F).

Our data suggest that blocking memory consolidating processes in the Te2 prevents changes in the BLA activity which may occur during memory retrieval processes. To better address this issue, we employed an alternative method based on the expression of the immediate early gene zif268. Immediate early genes are required for synaptic plasticity and are used as an index of neuronal activation (Frankland and Bontempi, 2005; Sacco and Sacchetti, 2010; Lesburguères et al., 2011; Kwon et al., 2012; Grosso et al., 2017). Among immediate-early genes, zif268 expression has been associated with long-term plasticity that occurs during memory retrieval (Frankland et al., 2004; Kwon et al., 2012; Xie et al., 2014; Grosso et al., 2017). Accordingly, several recent studies have shown changes in zif268 expression in the sensory cortex following emotional memory recall (Hall et al., 2001; Maviel et al., 2004; Sacco and Sacchetti, 2010; Kwon et al., 2012). By employing this technique, previous studies have shown that the activity of LA and central (CeA) amygdala nuclei is enhanced after the retrieval of long-term fearful memories (Sacco and Sacchetti, 2010; Kwon et al., 2012). Therefore, we tracked the expression of zif268 proteins induced in the LA, BA and CeA due to recall of fearful memories in unpaired group (n = 12), conditioned animals (n = 14) and in animals that received TTX in Te2 after training (n = 12; Figures 5A–C). In LA, memory recall produces an increase in zif268 protein levels in conditioned subjects compared to unpaired ones but not in TTX-treated rats (LA, unpaired, 26.82 ± 1.82; conditioned, 33.51 ± 2.21; TTX-Te2, 24.81 ± 1.12; F_(2,37) = 6.50, P = 0.004). Remarkably, the TTX-treated group did not differ from the unpaired one (Newman-Keuls test, P > 0.05; Figures 5A,D). These data suggest that LA processes require information processed early on in the cortex. No differences were detected among conditioned, TTX-treated and unpaired groups in zif268 protein expression level in the BA (BA, unpaired, 10.14 ± 0.61; conditioned, 10.00 ± 1.06; TTX-Te2, 9.59 ± 0.70; F_(2,37) = 0.10; P = 0.897; Figures 5B,E). The similarity between naïve and conditioned animals suggests that BA may be not involved in the conditioned freezing response, in line with previous studies showing that blockade of this site did not affect conditioned freezing to auditory CSs (Killcross et al., 1997; Amorapanth et al., 2000; Herry et al., 2008). Conversely, this nucleus may be more prominently required for the active avoidance of threats (Killcross et al., 1997; Amorapanth et al., 2000; Herry et al., 2008).

We finally repeated the analysis of zif268 expression in the CeA of unpaired, conditioned and TTX-injected rats. We found that an increase in zif268 protein levels in conditioned subjects compared to unpaired ones, but not in TTX-treated rats (CeA, unpaired, 0.92 ± 0.19; conditioned, 1.91 ± 0.24; TTX-Te2, 1.21 ± 0.19; F_(2,37) = 5.68, P = 0.005). Again, the TTX-conditioned group did not differ from the unpaired one (Newman-Keuls test, P > 0.05; Figures 5C,F).

Taken together, these results show that the reversible blockade of Te2 cortex during memory consolidation processes prevents the enhancement of zif268 expression that normally occurs in most individual amygdala nuclei during memory retrieval.